First, abbreviations used in this specification will be described.    aPL: anti-phospholipid antibody    aCL: anti-cardiolipin antibody    APS: antiphospholipid syndrome    β2-GPI: β2-glycoprotein I    β2-GPI/oxLDL complex: complex consisting of oxLDL and β2-GPI    BSA: bovine serum albumin    CL: cardiolipin    Cu2+-oxLDL: oxLDL oxidized with CuSO4     DEAE: diethylaminoethyl    EDTA: ethylenediaminetetraacetic acid    ELISA: enzyme-linked immunosorbent assay    HRP: horseradish peroxidase    LDL: low-density lipoprotein    MDA: malondialdehyde    OD: optical density    oxLDL: oxidized LDL    oxLig-1: 7-ketocholesteryl-9-carboxynonanoate;    9-oxo-9-(7-ketocholest-5-en-3β-yloxy)nonanoic acid (IUPAC)    oxLig-2: 7-ketocholesteryl-12-carboxy(keto)dodecanoate    PAPS: primary antiphospholipid syndrome    PBS: phosphate buffered saline    PL (PLs): phospholipid(s)    SLE: systemic lupus erythematosus    TBARS: thiobarbituric acid reactive substance    TLC: thin layer chromatography    9-COOH-22KC: 7-ketocholesteryl-13-carboxytridecanoate;    13-oxo-13-(7-ketocholest-5-en-3β-yloxy)tridecanoic acid
It is known that β2-GPI is a major antigen which is recognized by “phospholipid antibody” occurring in patients with APS, and binds specifically to oxLDL but not to non-oxidized (native) LDL. WO 95/9363 discloses a method of measuring oxLDL by using such ability of β2-GPI to bind specifically to oxLDL, its applied kit for diagnosis of arteriosclerotic diseases, etc. Further, J. Lipid Res., 42, pp. 697-709 (2001) [reference 7] discloses that β2-GPI recognizes the structural part of oxLig-1 in oxLDL and binds thereto.
oxLDL forms a complex with β2-GPI (β2-GPI/oxLDL complex) in the living body, and by measuring this complex occurring in the living body, various diseases can be detected. As a standard for measuring this complex occurring in the living body, Cu2+-oxLDL and β2-GPI which were contacted with each other without pre-incubation have been conventionally used.
A standard for measuring the hardly dissociable β2-GPI/oxLDL complex occurring in the living body is desirably the same as, or as similar as possible to, the standard for measuring the complex occurring in the living body. However, a mode of bonding between oxLDL and β2-GPI, etc., in the β2-GPI/oxLDL complex occurring in the living body and in the β2-GPI/oxLDL complex used conventionally as the standard have not been known.